This invention relates to the fields of molecular biology and neoplastic disease, and more specifically, to isolated nucleic acids, proteins, antibodies, methods and kits containing the same which are useful in genetic screening assays, and in the design of clinically beneficial chemotherapeutic agents which inhibit the aberrant cellular proliferation in tumor cells.
Several publications are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Pancreatic cancer is the fifth leading cause of death by cancer in the United States. Twenty-four thousand people die each year from this disease. The 5-year survival for pancreatic cancer patients is less than 5% and the incidence of the disease has tripled over the last 40 years. The molecular basis underlying the pathogenesis of pancreatic adenocarcinoma remains unknown. As a result,:the disease has an extremely poor prognosis and lacks early diagnostic and therapeutic modalities.
Normal cellular proliferation is finely regulated by the expression of growth-promoting proto-oncogenes and growth-controlling anti-oncogenes. Mutations, rearrangements, deletions, or amplifications that potentiate the activities of proto-oncogenes result in tumor formation. Similar events that inactivate anti-oncogenes or tumor suppressor genes disrupt their role in the cell as negative regulators of cell growth and proliferation.
Gene amplification has been implicated as a common mechanism by which tumor cells acquire a chemotherapy resistant phenotype. Some amplification units arise at the site of the normal gene, but disperse into the cytoplasm as double minutes (DMs). These DMs may become reincorporated and reamplified as homogeneously staining regions (HSRs) or abnormal banding regions (ABRs) at other sites in the genome. DMs and HSRs may be alternate forms of amplified DNA. The DMs are not inherited stably during cell division because of the lack of centromeres. Integration of DMs into a chromosome is thought to result in the formation of HSRs, which represent a more stable, form of the amplified DNA which is maintained as the cell divides. The mechanisms underlying this process are not completely understood, but appear to be based on recombination and unequal distribution of the amplified DNA into daughter cells.
Cytogenetic amplification has been observed in 8 of 63 primary pancreatic adenocarcinomas analyzed for the presence of DMs. The expression of epidermal growth factor, epidermal growth factor receptor, transforming growth factor, erbB-2, erbB-3, and c-met are elevated in pancreatic cancer (Barton et al., 1991; Korc et al., 1992; Lemoine et al., 1992; Prat et al., 1991).
Using restriction landmark genomic scanning (RLGS), Miwa et al (1996) identified a locus at chromosome 19ql3.1-ql3.2 including the AKT2 gene which was amplified in 20% of pancreatic cancer. Over expression of the AKT2 gene was further shown to be associated with the malignant phenotype of a subset of human ductal pancreatic cancers (Cheng et al., 1996).
No well-defined differentiation pathway has been shown in pancreatic adenocarcinomas, and the biology of this tumor type is generally poorly understood. The present inventors have appreciated a need for the isolation of essential components involved in the regulation of differentiation and proliferation of pancreatic tumor cells. Molecular elucidation of these components will provide novel targets for the development of antiproliferative and diagnostic agents for cancer treatment and diagnosis.
This invention provides novel, biological molecules useful for identification, detection, and/or molecular characterization of components involved in the regulation of cellular differentiation and tumorigenesis. According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes an isolated open reading frame encoding a phosphoprotein of a size between about 60 to 70 kilodaltons. The encoded protein, referred to herein as PD2, comprises a tripartite domain structure including a nuclear transport signal, a helix-loop-helix domain, a nucleotide binding site, and several putative phosphorylation sites.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a DNA encoding a human PD2 protein. In a particularly preferred embodiment, the human PD2 protein has an amino acid sequence the same as SEQ ID NO:2. An exemplary PD2 nucleic acid molecule of the invention comprises SEQ ID NO:1.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) SEQ ID NO:1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of SEQ ID NO:1; (3) a sequence comprising preselected portions of SEQ ID NO:1, (4) a sequence encoding part or all of a polypeptide having amino acid SEQ ID NO:2. Such partial sequences are useful as probes to identify and isolate homologues of the PD2 gene of the invention. Accordingly, isolated nucleic acid sequences encoding natural allelic variants of the nucleic acids of SEQ ID NO:1 are also contemplated to be within the scope of the present invention. The term natural allelic variants will be defined hereinbelow.
According to another aspect of the present invention, isolated human PD2 protein is provided. PD2 is a phosphoprotein with a deduced molecular weight of between about 60 kDa and 70 kDa. PD2 comprises a tripartite domain structure including a nuclear transport signal, a helix-loop-helix domain, a cyclic AMP or related nucleotide binding site, and several putative phosphorylation sites. The expression of this PD2 protein correlates with the deregulated growth of highly undifferentiated pancreatic adenocarcinomas.
In a preferred embodiment of the invention, the protein is of human origin, and has the amino acid sequence of SEQ ID NO:2. In a further embodiment the protein may be encoded by natural allelic variants of SEQ ID NO:1. Inasmuch as certain amino acid variations may be present in human PD2 protein encoded by natural allelic variants, such proteins are also contemplated to be within the scope of the invention.
According to another aspect of the present invention, antibodies immunologically specific for the human PD2 protein described hereinabove are provided.
Host cells comprising the PD2 encoding nucleic acids of the invention are also contemplated to be within the scope of the present invention. Such host cells include but are not limited to bacterial cells, mammalian cells, insect cells, fungal cells, and plant cells. The PD2- encoding nucleic acids may be conveniently cloned into a plasmid or retroviral vector for introduction into host cells. Such cells are useful in screening methods to identify compounds which regulate and/or inhibit PD2 expression. Compounds so identified may have therapeutic value in the treatment of pancreatic cancer.
The present invention also encompasses transgenic mice expressing the PD2 encoding nucleic acids of the invention. The PD2 encoding DNA may be altered to include any of the following, mutations, alterations, deletions, insertions. In another embodiment, PD2 knockout mice may be generated to assess the contribution of the PD2 gene to growth and development.
This invention also provides methods for genetic screening and diagnostic evaluation of patients at risk for, or currently suffering from, cancer of the pancreas. The hybridization specificity of the nucleic acids of the invention may be used for differential evaluation of patients presenting with phenotypic characteristics common to pancreatic cancer. The nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for PD2 or mutations thereof. Additionally, antisense molecules which may be useful in the regulation of PD2 expression are provided herein. Other methods encompassed by the present invention include immunodetection methods for assessing biological samples for the presence of PD2 proteins.
In another aspect of the present invention, kits are provided for practicing the methods set forth above. An exemplary kit for screening tumor samples for PD2 expression includes for example, suitable primers for PCR amplification of target PD2 sequences. Exemplary primers include those having the sequence of SEQ ID NOS: 12 and 13. A kit in accordance with the invention may also contain vials, buffers, a target PD2 sequence as a positive control and a protocol sheet. Another exemplary kit may employ immunological methodology. Kits of this type include immobilized PD2 protein and antibodies immunologically specific for PD2. Such kits may be used in for immunological assessment of biopsy specimens for identification and/or quantification of PD2 in pancreatic tissues.
The term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used with reference to nucleic acids of the invention. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5xe2x80x2 and 3xe2x80x2 directions) in the naturally occurring genome of the organism from which it originates. For example, the xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
When used with reference to RNA molecules of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a substantially pure form.
The terms xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d are sometimes used herein to refer to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, these terms may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in substantially pure form.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to antibodies of the invention, the term xe2x80x9cimmunologically specificxe2x80x9d refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., PD2), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to nucleic acids and oligonucleotides, the term xe2x80x9cspecifically hybridizingxe2x80x9d refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). When used in reference to a double stranded nucleic acid, this term is intended to signify that the double stranded nucleic acid has been subjected to denaturing conditions, as is well known to those of skill in the art. In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The nucleic acids, proteins, and antibodies, of the present invention may be used to advantage diagnostic reagents and tools for assessing the malignant potential of pancreatic adenocarcinomas. They may also be used as targets for the development of novel chemotherapeutic agents that regulate differentiation and/or inhibit aberrant cellular proliferation in tumor cells. The transgenic mice of the invention provide a means to assess the function of PD2 in vivo.
The human PD2 molecules described are above may also be used as research tools and will facilitate the elucidation of the genetic and protein interactions involved in the regulation of cell division, differentiation, and neoplastic transformation. Methods and kits employing such molecules are also provided.